Venous thrombosis (VT) affects over 1 million Americans annually. Even with treatment, 30-50% of VT patients develops recurrent VT or suffer debilitating long-term morbidity including chronic pain, edema, and ulcers (so- called post-thrombotic syndrome). Current anticoagulation therapies target factor Xa or thrombin and are associated with a significant risk of catastrophic bleeding. The development of next generation agents which reduce thrombosis without increasing the risk of hemorrhage, would have a profound impact on this significant clinical problem. The defining characteristics of venous red thrombi are their high red blood cell (RBC) and fibrin content. The existing paradigm suggests RBCs are passively trapped in the clot during fibrin polymer formation. However, we have recently discovered that factor XIII (FXIII) mediates RBC retention in clots and determines clot size. We have also identified fibrinogen residues that promote interactions with FXIII and are required for normal FXIII activation and fibrin crosslinking. Importantly, we have shown that inhibiting FXIIIa activity or blocking FXIII-fibrinogen interactions reduces clot RBC content, and consequently, significantly reduces thrombus size in vitro and in vivo. These exciting discoveries suggest that inhibiting FXIII or blocking FXIII-fibrinogen interactions represent entirely new and novel strategies for reducing VT. The goal of this application is to determine the molecular mechanisms by which FXIII and fibrinogen mediate VT. The overall hypothesis is that FXIII(a) activity mediates RBC retention in clots. Thus, inhibiting FXIII activiy or disrupting the interaction between FXIII and fibrinogen will reduce VT. This hypothesis will be tested in three Specific Aims: 1) Define the fibrinogen residues required for FXIII binding and determine whether peptides that disrupt FXIII interactions with fibrinogen decrease RBC retention in clots, 2) Determine whether FXIIIa promotes RBC retention in clots via fibrin crosslinking, and 3) Determine the role of FXIII and effect of FXIII(a) inhibition in VT and hemostasis. This study will employ biochemical, genetic and pharmacologic tools to define the FXIII- fibrinogen axis and determine the role of FXIII activity and RBC retention during whole blood clot formation. FXIII-fibrinogen interactions and FXIIIa function will be examined using recombinant proteins, solution phase binding assays, and innovative fibrin analytical methods. Clot formation will be studied using in vitro and in vivo models of VT and hemostasis, including live-animal imaging (e.g., ultrasound imaging and intravital microscopy). These studies will significantly expand our understanding of newly-recognized roles for FXIII(a) and fibrinogen in thrombus formation and stability. This study is highly innovative because it challenges the current paradigm that RBCs are passively entangled in thrombi and proposes that thrombus RBC content can be reduced to prevent VT. The proposed research is clinically significant because it may reveal an entirely new strategy to reduce VT.